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Home , Chromoblastomycosis, Coccidioides posadasii, Dermatophytosis, Eumycetoma, Favus, Fusariosis

REVIEW

Immunity against fungi

Michail S. Lionakis,1 Iliyan D. Iliev,2 and Tobias M. Hohl3 1Fungal Unit, Laboratory of Clinical Infectious , National Institute of and Infectious Diseases, NIH, Bethesda, Maryland, USA. 2Jill Roberts Institute for Research in IBD, Department of Medicine, Weill Cornell Medical College, , New York, USA. 3Infectious Service, Department of Medicine, and Immunology Program, Memorial Sloan Kettering Center, New York, New York, USA.

Pathogenic fungi cause a wide range of syndromes in immune-competent and immune- compromised individuals, with life-threatening disease primarily seen in with HIV/AIDS and in patients receiving immunosuppressive therapies for cancer, autoimmunity, and end-organ failure. The discovery that specific primary immune deficiencies manifest with fungal and the development of models of mucosal and invasive mycoses have facilitated insight into -specific recognition, signaling, effector pathways, and adaptive immune responses. Progress in deciphering the molecular and cellular basis of immunity against fungi is guiding preclinical studies into vaccine and immune reconstitution strategies for vulnerable patient groups. Furthermore, recent work has begun to address the role of endogenous fungal communities in health and disease. In this review, we summarize a contemporary understanding of protective immunity against fungi.

Introduction Humans are exposed to fungi throughout life via inhalation, digestion, and/or traumatic inoculation of fungal particles. The vast majority of these encounters are asymptomatic, and less than 100 of the estimat- ed 5 million fungi are associated with human disease (1) (Table 1 and Figure 1). Fungi can either exist as spherical cells (e.g., Cryptoccocus neoformans) or as that form branching tubular hyphae (e.g., fumigatus). Dimorphic fungi (e.g., capsulatum) grow as molds in the environ- ment and in human tissue. grows as yeast cells and pseudohyphae, a hyphal form with tapered ends, in human tissue; this morphologic switch is essential for virulence (2). Fungi were recognized to cause disease during investigations into the scalp , which was widespread in 19th century (3). German physiologist Robert Remak (1815–1865) immersed favus samples in acetic acid and observed fungal hyphae and conidia (named schönleinii in honor of Johann Schönlein, Remak’s mentor). In 1842, Remak injected favus crust–isolated material into his forearm and noted growth in the lesions, thereby establishing causality between the fungus and disease. Several events, including the advent of myeloablative chemotherapy for neoplasia, and immune modulators for autoimmunity, transplantation for end-organ failure, and the AIDS pan- demic, contributed to the emergence of fungal infections in the second half of the 20th century. Novel pathogenic fungi that pose a threat to humans (e.g., ), amphibians (e.g., Batrachochytri- um dendrobatidis), and bats (e.g., Pseudogymnoascus destructans) have also been identified (4). In response, research in fungal pathogenesis and immunity has intensified to inform vaccine- and thera- py-based approaches for mycoses (5). This review focuses on insights gained from animal models and patients with primary immune deficiency disorders (PIDDs), but does not cover allergenic or toxin- mediated fungal disease (6, 7).

Conflict of interest: The authors have Antifungal immunity from the bench: contribution of animal models declared that no conflict of interest This section focuses on antifungal immunity to different yeasts, molds, and dimorphic fungi. Contempo- exists. rary animal models of fungal infection are reviewed elsewhere (8). Published: June 2, 2017 Fungal recognition and immune activation. The fungal contains polysaccharide and lipid moi- Reference information: eties that activate immune responses (9) (Table 2). The cell wall is exterior to the plasma membrane and JCI Insight. 2017;2(11):e93156. https:// arranged in layers: the innermost layer typically consists of chitin, an N-acetylglucosamine polymer; the doi.org/10.1172/jci.insight.93156. adjacent external layer is formed by immunoreactive β-(1,3) and β-(1,6) glucans, which are concealed by insight.jci.org https://doi.org/10.1172/jci.insight.93156 1 REVIEW

many fungi. H. capsulatum employs an α-glucan layer and the action of a glucanase (9–11). A. fumigatus resting conidia utilize a proteinaceous layer (12), while the hyphal cell wall layer contains galactomannan and galactosaminogalactan, the latter of which conceals inflammatoryβ -glucan (13, 14). The C. albicans outer cell wall consists of glycoproteins that incorporate N- and O-linked mannans and induces inflammatory responses via the mannose receptor and TLR-4 (15).C . albicans mannans conceal β-glucans as well; the latter are exposed on bud and birth scars during yeast cell division (9). The C. neoformans capsule covers the chitinous and β-glucan–rich cell wall layers and largely consists of glucuronoxylomannan and galactoxylomannan (16). Previously published reviews provide in-depth discussion of fungal cell wall architecture (9, 16–18). At portals of entry fungal cells encounter and bind to antibodies, complement, and soluble pat- tern recognition receptors. Collectively, these interactions facilitate signaling responses by membrane- bound receptors and the induction of antifungal effector mechanisms (5, 9). In the , the collectin pentraxin-3 (PTX3) binds to A. fumigatus conidial galactomannan (19) and facilitates complement deposition and CD32-dependent conidial uptake by (20). Ptx3–/– mice are susceptible to respiratory A. fumigatus challenge (19), and a PTX3 polymorphism enhances the vulnerability of hema- topoietic cell transplant recipients to invasive (21). The C- lectin receptor (CLR) dectin-1 (encoded by Clec7a) binds β-glucans from a variety of fungi, including those on C. albicans bud scars and germinating A. fumigatus conidia, and activates sig- naling responses to Pneumocystis jiroveci, H. capsulatum, posadasii, and Paracoccidioides brasil- iensis (5, 9, 22–24) (Figure 2). β-Glucan binding displaces regulatory phosphatases CD45 and CD148 (25), induces SRC-dependent phosphorylation of the intracellular ITAM-like motif, and recruits the SHP-2 phosphatase (26). SYK docks to this scaffold and transduces signals via PKC-δ (27) and the VAV of GEFs (28) to CARD9, which complexes with BCL10 and MALT1 (29) to activate the canonical NF-κB subunits p65 and c-REL (30). Dectin-1 signaling also modulates the noncanonical NF-κB subunit RELB through RAF-1–dependent phosphorylation and deacetylation (31). In macro- phages and dendritic cells (DCs), the CARD9/BCL10/MALT1 complex directs Il1b transcription and caspase-1– and caspase-8–dependent IL-1β release (32, 33), in part via the activity of NRLP3- and AIM2-containing inflammasomes (34). Rubicon can disrupt signal transduction and NF-κB activation via the CARD9/BCL10/MALT1 complex (35). Dectin-1/SYK/CARD9–dependent , such as TNF, CXCL2, IL-6, IL-23, and IL-1β, promote innate immune activation and Th17 differentiation (36). In addition, dectin-1/SYK signaling in DCs induces IFN-β production via IRF5 (37). The role of type I IFN signaling in defense against remains controversial, with both protective (37) and detrimental (38) phenotypes reported. Dectin-1 signaling regulates ERK (also known as MAP kinase) activity via H-RAS and RAS guanine nucleotide–releasing factor 1 (RASGRF1) (39). This pathway regulates IL-6, IL-1β, and TNF, but not IL-12 responses, and is protective during systemic candidiasis (39). c-JUN kinase isoform 1–defi- cient (JNK1-deficient) mice are resistant to systemic candidiasis (40). Dectin-1–induced JNK1 signaling negatively regulates CD23 (encoded by Fcer2) expression via nuclear factor of activated T cells (NFAT) activation. CD23 binds α-mannans and β-glucans and induces the antifungal effector NOS2. Consistent with this model, Cd23–/– mice are susceptible to systemic candidiasis (40). In otherwise nonphagocytic cells, dectin-1 expression promotes phagocytosis of nonopsonized β-glucan particles (5, 9). Bruton’s tyrosine kinase (BTK) and VAV-1 interact with dectin-1 in during C. albicans phagocytosis, a process impaired by genetic loss of either (41). Dectin-1/SYK/ CARD9 signaling in NADPH oxidase activity is controversial, as dectin-1–dependent (24) and –indepen- dent (42) control of β2 integrin (CD18) activation and the respiratory burst have been reported in vitro. Murine Clec7a –/– and Card9–/– neutrophils display no cell-intrinsic defect in killing A. fumigatus conidia, unlike p47phox–/– neutrophils (43). These data can be reconciled if the major role of dectin-1/CARD9 is to modulate NADPH oxidase and fungal killing via soluble mediators, rather than by cell-intrinsic activa- tion. β2 integrins and TLR signaling can collaborate with dectin-1 to mount macrophage inflammatory responses to H. capsulatum and other fungi (44, 45). Dectin-2 (encoded by Clec4n) forms a complex with dectin-3 (encoded by Clec4d) to bind Candida α-mannans (46, 47) or with mincle (encoded by Clec4e) to bind glycolipids (48). Blastomyces der- matiditis, H. capsulatum, C. posadasii, and A. fumigatus induce dectin-2 signaling (43, 49). As these CLRs lack a signaling , heterodimeric complexes signal via the ITAM-coupled adaptor FcRγ (9). insight.jci.org https://doi.org/10.1172/jci.insight.93156 2 REVIEW

Table 1. Common human fungal diseases and associated PIDDs

Mycosis Morphotype Clinical Syndromes (syndromes) linked to enhanced Fungal species susceptibilityA Most common genera Aspergillosis and systemic disease in immune-compromised NADPH oxidase (CGD); STAT3 (HIES); Aspergillus spp., A. hosts; allergenic disease (e.g., ABPA) in atopic hosts; cavitary CARD9 (extrapulmonary aspergillosis); fumigatus, A. flavus, A. niger, disease in setting of structural lung disease; deep tissue GATA2 (MonoMAC syndrome); CD18 A. terreus, A. nidulans (in organ disease (e.g., abscess, osteomyelitis) in CGD and (LAD); ELA2, HAX1 (SCN) CGD) CARD9-deficient patients Mold Pneumonia, sinus, and systemic disease in immune- , and compromised hosts and in spp. Mold Pneumonia, cutaneous, bloodstream, and systemic disease in NADPH oxidase (CGD), STAT1 GOF oxysporum and F. immune-compromised hosts; mutations solani Mold Skin and infections (i.e., keratinized tissues), e.g., tinea NADPH oxidase (CGD); CARD9 (deep , pedis (athlete’s foot), ringworm tissue infections) Trichophyton, spp. Dimorph Lymphocutaneous disease, ascending lymphangitis NADPH oxidase (CGD) Sporothrix schenkii Dimorph Pneumonia; skin, mucosal, skeletal, and genitourinary GATA2 (MonoMAC syndrome) Blastomyces dermatiditis disease Dimorph Pneumonia, mediastinal and fibrosis, IL12RB1; IFNGR1; STAT1 GOF H. capsulatum (North (intracellular in disseminated histoplasmosis mutations; STAT3 (HIES); UNC119, America), H. dubousii (Africa) macrophages) MAGT1, RAG1 (idiopathic CD4 lymphopenia); DOCK8 (HIES); CD40 ligand Dimorph Pneumonia, skeletal disease, IL12RB1; IFNGR1; STAT1 GOF C. immitis, C. posadasii mutations; STAT3 (HIES) Dimorph Pneumonia; skin, mucosal, and skeletal disease IL12RB1 P. brasiliensis Penicilliosis Dimorph Pulmonary, skin, mucosal, and disseminated disease marneffei Trophozoites Pneumonia; common AIDS-defining illness CD40 ligand, >30 genes that underlie P. jiroveci (asexual) and SCID; MHC class II deficiency; NEMO; cysts (sexual) DOCK8 (HIES); CARD11; Wiskott- Aldrich syndrome Candidiasis Dimorph Thrush, vulvovaginitis; bloodstream and systemic infections CMC: IL17F; IL17RA; IL17RC; ACT1; C. albicans, C. tropicalis Yeast in immune-compromised patients; AIDS-defining illness; STK4; IRF8; CARD9; STAT1 GOF C. glabrata, C. krusei, deep tissue single organ disease (e.g., kidney or , mutations; STAT3 (HIES); RORC; AIRE; C. parapsilosis, C. auris osteomyelitis) in CGD and other PIDDs; deep tissue disease anti–IL-17 autoantibodies (thymoma) uncommon in syndromes associated with CMC with the CNS disease: CARD9 exception of CARD9 deficiency Invasive and deep tissue disease: ELA2, HAX1 (SCN), CGD, complete MPO deficiency Yeast Pneumonia, meningitis; AIDS-defining illness Anti–GM-CSF or –IFN-γ antibodies; C. neoformans, C. gattii (pigmented, CD40 ligand; GATA2; IL12R; STAT3 encapsulated) (HIES) Yeast Chronic infection of cutaneous and subcutaneous tissues Fonsecaea pedrosii, and (pigmented) others Molds (pale or Chronic infection of cutaneous and subcutaneous tissues; spp., Madurella dark grains) Scedosporium spp. associated with bloodstream, pulmonary spp., and others and CNS disease in immune-compromised hosts AWe did not discuss all listed defects associated with mycoses in the text. The reader is referred to references 64 and 65 for a more comprehensive discussion and additional links to the primary literature. ABPA, allergic bronchopulmonary aspergillosis; CARD9, caspase recruitment domain–containing protein 9; CMC, chronic mucocutaneous candidiasis; CGD, chronic granulomatous disease; CNS, central nervous system; DOCK8, dedicator of cytokinesis 8; GM-CSF, granulocyte-macrophage colony-stimulating factor; GATA2, GATA-binding protein 2; GOF, gain of function; HIES; hyper-IgE syndrome; LAD; leukocyte adhesion deficiency; NEMO, NF-κB essential modulator; PIDDs, primary immune deficiency disorders; SCID; severe combined ; SCN; severe combined ; STAT, signal transducer and activator of transcription.

insight.jci.org https://doi.org/10.1172/jci.insight.93156 3 REVIEW

Figure 1. Human fungal diseases. The figure depicts the anatomic sites that are most commonly affected by the fungal genera listed below each organ system. At several sites, fungal disease occurs due to inoculation at the site. For example, humans inhale infectious conidia or desiccated fungal cells of all genera listed under and respiratory tree. For other sites, such as the , , and blood stream, disease is the result of dissemination of the indicated fungus from the initial inoculation site. Illustrated by Mao Miyamoto.

Following fungal recognition, the E3 ubiquitin ligase CBLB ubiquitinates dectin-1, dectin-2, and SYK, targeting them for degradation (50–52). In systemic candidiasis, Cblb–/– mice exhibit reduced renal fungal burden and improved survival due to decreased inflammation-driven tissue damage (50–52). In contrast, TRIM62-mediated CARD9 ubiquitination is essential for BCL10 interactions, NF-κB activation, and defense against candidiasis (53). Mincle/SYK/CARD9 signaling mediates responses to Fonsecaea pedrosii, an agent of chromoblas- tomycosis (54). Mincle-induced cytokines are insufficient to clear this soft tissue infection, and applica- tion of TLR-7 agonist imiquinod to fungal lesions can restore sterilizing immunity (55). In DCs, mincle signaling activates the E3 ubiquitin ligase MDM2, in turn suppressing dectin-1– and IRF1-dependent Il12a transcription (56). This MDM2-dependent pathway impairs Th1 responses and may contribute to the chronicity of chromoblastomycosis. During murine pneumocystosis and candidiasis, mincle signal- ing enhances fungal clearance but is dispensable for survival (57, 58). Loss of individual CLRs results in variable susceptibility to C. albicans (22, 23) and A. fumigatus (24, 43), in part due to strain-specific differences in CLR activation, as shown forC . albicans (59). Consistent with SYK/CARD9 being central for CLR signal integration, mice with hematopoietic or DC-specific Syk deletion or with global CARD9 deficiency are highly susceptible toC . albicans (29, 60) and A. fumig- atus (43) challenge. CARD9/SYK–dependent susceptibility maps to defective responses that control , NK, and T cell trafficking or activation. During systemic candidiasis, DCs secrete IL-23 in a SYK-dependent manner, prompting IL-17A–dependent (hereafter referred to as IL-17) NK insight.jci.org https://doi.org/10.1172/jci.insight.93156 4 REVIEW

Table 2. Mammalian signaling receptors and cognate fungal ligands and species

C-type lectin receptors: Fungal ligand Fungal speciesA ReferencesB Dectin-1 (Clec7a) β-Glucans AF, CA, CP, ER, HC, PB, PC (23, 24, 44, 49, 59, 184) Dectin-2 (Clec4n) α-Mannans, O-linked mannoproteins AF, BD, CA, CP, CG (48, 49, 97, 185) Dectin-3 (CLECSF8, MCL, Clec4d)C α-Mannans CA (47, 186) Mincle (Clec4e) α-Mannosyl residues, glyceroglycolipids CA, FP, MS, PC (48, 54, 58) CD209 (DC-SIGN) Galactomannan, mannans AF, CA (187) Mannose Receptor N-linked mannans, mannans CA, PC (15, 188) Toll-like receptors: TLR-2 α-(1,4)-Glucans CA (189) TLR-1 and TLR-2 Glucuronoxylomannans CN (190) TLR-2–TLR-6 Phospholipomannans, glucuronoxylomannans CA (191) CN TLR-4 O-linked mannans, rhamnomannans CA, SA (15, 189, 192) TLR-9 Unmethylated DNA AF, CA (63) NOD-like receptors: NOD1 Unknown AF (193) NOD2 Chitin (?) CA (194) NLRC4 Unknown CA (195) NLRP3 Unknown AF, CA (34, 196) Other receptors: CD14 α-(1,4)-Glucans SA (197) CD23 (Fcer2a) α-Mannan, β-glucan CA (40) CD36 CN, CA (198)

CR3 (integrin α2/βM; CD11b/CD18; Mac-1) β-Glucan AF, CA, HC (42, 44, 45, 72, 73) Galectin-3 β-Mannosides CA (199) Lactosylceramide (glycosphingolipid) PC (200) Pentraxin-3 (soluble) Galactomannan AF (19, 62) ARole in murine survival or fungal clearance. BThe references focus on manuscripts that demonstrate in vivo roles for receptors in murine survival or fungal clearance; additional references to the primary literature can be found in references 5 and 9. CDectin-3 can form heterodimeric complexes with dectin-2 or with mincle. AF, ; BD, Blastomyces dermatiditis; CA, Candida albicans; CG, ; CN, , CP, ; ER, Exserohilum rostratum; FP, Fonsecaea pedroisi; HC, ; MS, Malassezia spp.; PB, Paracoccidioides brasiliensis; PC, Pneumocystis carinii from sp. muris; SA, Scedosporium apiospermium.

cell release of GM-CSF, thereby activating candidacidal neutrophils at infection sites (60, 61). CLR-independent pathways also mediate fungus-induced inflammation. Fungal ligands activate TLR1–4, TLR-6, TLR-9, and NOD2 signaling in a variety of cell types (5, 9). For example, PTX3-opso- nized A. fumigatus conidia activate TLR-4/MD-2/TRIF–dependent signaling that mediates IL-10 produc- tion (62). Following internalization, A. fumigatus conidia trigger macrophage TLR-9–, calcineurin-, and BTK-dependent TNF release (63); however, humans with NLR or TLR/MyD88 signaling defects do not manifest with fungal infections (64, 65). A detailed discussion of Mendelian defects that do manifest with fungal infections follows later in this review. Fungal killing. Neutrophils rapidly internalize conidia and/or yeast cells and direct the production of ROS to the fungal phagosome (66). In macrophages, NADPH oxidase–dependent LC3-associated phago- cytosis represents a potential clearance mechanism for A. fumigatus conidia (67, 68). Nutritional immunity, exemplified by zinc and iron sequestration, is critical for macrophage defense against intracellularH . cap- sulatum yeast cells (69), against fungal keratitis (70), and in chronic granulomatous disease (CGD) patients with defects in NADPH oxidase (71–73). Fungal hyphae (A. fumigatus) and pseudohyphae (C. albicans) induce NADPH oxidase–dependent neutro- phil extracellular trap (NET) formation (74), a form of neutrophil programmed cell death termed NETosis. NETs contain extracellular nucleic acids, histones, and granular , including calprotectin and PTX3 (75, 76), and ensnare fungal that are too large for phagolysosomal killing (74). Neutrophil calpro- tectin, a major NET component, is important for antihyphal host defense during murine A. fumigatus keratitis yet dispensable for anticonidial defense following respiratory A. fumigatus challenge (77), illustrating a fungal insight.jci.org https://doi.org/10.1172/jci.insight.93156 5 REVIEW

Figure 2. Model of fungus-induced CLR signaling in antifungal defense. At the site of inoculation, particulate fungal polysaccharides bind C-type lectin receptors (CLRs) and Fc receptors (FcRs), resulting in SYK activation via ITAM signaling in the receptor tail, the FcRγ signaling adaptor, or integrin receptor activation. The ensuing PKCδ and CARD9 activation is critical for caspase-1 and caspase-8 activity, MAP kinase signaling (not shown), NF-κB activation, and cytokine production. SYK-dependent PLCγ2 activation is linked to NADPH oxidase assembly and calcineurin-dependent NFAT activation. Dectin/SYK signal- ing controls IRF5-dependent IFN-γ production. VAV1 and BTK can interact with dectin-1 to mediate phagocytosis of Candida albicans in macrophages. BTK may also promote calcineurin activation and the production of NFAT-regulated cytokines. The model depicts fungal killing in the phagosome. Myeloid cell– derived cytokines promote the differentiation of both Th1 cells and Th17 cells. Th17 and innately derived IL-17 activate epithelial responses that coordinate clearance of fungal cells at mucosal surfaces. The asterisks indicate genetic or humoral defects that predispose humans to a spectrum of fungal disease. Myeloid and epithelial cells collaborate to regulate CXCR2-dependent neutrophil recruitment to portals of infection. Illustrated by Mao Miyamoto.

morphotype-specific role in host defense.A . fumigatus and galactosaminogalactan enhance fun- gal resistance to NETs (78, 79). The contribution of NETs to fungal clearance in vivo remains difficult to quan- tify, because researchers lack experimental tools that specifically disrupt NETosis in murine models. Mucosal defense and fungal tissue invasion. At mucosal surfaces, IL-17 is critical for antifungal immunity. Innate lymphoid, Th17, and γδ T cells produce IL-17 following C. albicans challenge in the oropharynx or skin (80, 81). Skin sensory neurons participate in C. albicans detection and activate dermal DCs via the neuropeptide calcitonin-related gene product to direct IL-23–dependent γδ T cell IL-17 release (82). Oral epithelial cells represent an important IL-17 signaling target, as mice lacking IL-17–sensing capacity in these cells display a phenotype similar to that of Il17ra–/– mice (83). The IL-17–dependent transcriptional response of oral epithelial cells includes peptides, including β-defensin 3 (DEFB3). Accord- ingly, Defb3–/– mice are susceptible to oropharyngeal candidiasis (83). In C. albicans–infected kidneys, IL-17– dependent responses include activation of the kallikrein/kinin system, which prevents apoptosis of tubular cells (84). Neutrophil recruitment to infected oral tissues is complex, with reports of IL-17–dependent and –independent trafficking pathways (85, 86). One IL-17–independent mechanism involves IL-1α/β–sensing oral keratinocytes that regulate neutrophil influx via CXC-chemokine release and indirect control over G-CSF–dependent granulopoiesis (87). Thus, mucosal infections induce crosstalk between epithelial and hematopoietic cells to regulate innate activation and fungal clearance. insight.jci.org https://doi.org/10.1172/jci.insight.93156 6 REVIEW

Fungal invasion of epithelial and endothelial cells contributes to tissue damage and disease dissemina- tion. During mucormycosis, the endothelial receptor -regulated protein 78 (GRP78) enables Rhizo- pus oryzae hyphae binding to endothelial cells (88) via coat protein surface proteins (CotH), primar- ily CotH3. Metabolic changes associated with hyperglycemia and diabetic ketoacidosis (DKA) enhance endothelial GRP78 expression, resulting in GRP78-dependent fungal tissue invasion (89). The widespread expression of CotH family members among and absence from other fungal may explain the unique susceptibility of patients with DKA to mucormycosis. Consistent with this model, anti- bodies directed against GRP78 or CotH3 protect mice from mucormycosis (90). The C. albicans adhesin Als3 mediates binding to biotic and abiotic surfaces, including epithelial and endothelial cells, and triggers internalization via E- and N-cadherin (91). The adhesin Hwp1 and the heat-shock protein Ssa1 also contribute to this process (92). An Als3p-based vaccine has emerged as a promising candidate to prevent mucosal and systemic disease (93) (NIH ClinicalTrials.gov identifiers: NCT01926028, NCT02996448). Following epithelial attachment, C. albicans pseudohyphae secrete the pore-forming, cytolytic peptide toxin candidalysin that is essential for virulence (94). At the onset of epi- thelial invasion, candidalysin activates MAPK signaling and c-FOS activation at sublytic concentrations (94). Epithelial activation leads to IL-1α, IL-6, G-CSF, and GM-CSF release, alerting host cells to the presence of invasive pseudohyphae. A. fumigatus conidia express the lung mucin–binding lectin FleA that promotes lung macrophages to internalize conidia (95). Mice challenged with ΔfleA conidia have more severe pneumonia than challenged with WT conidia. Thus, FleA represents an essential target of the to clear inhaled conidia. A. fumigatus hyphae also invade epithelial and endothelial cells, a property mediated in part by the CalA protein (96) and by galactosaminogalactan (14). Although CalA is dispensable for host cell adherence, CalA stimulates integrin α5β1–dependent hyphal endocytosis and is required for virulence in immunosuppressed mice (96). Trained immunity and fungal infections. Humans maintain sterilizing antifungal immunity in the lung despite daily inhalation of thousands of fungal cells; therefore, single-inoculum animal models are limited in revealing immunologic responses that arise in response to multiple challenges. Repeated exposure to A. fumigatus antigens gives rise to RORγt+IL-17+ neutrophils in response to IL-6 and IL-23 production (97). IL-17+ neutrophils express IL-17RC and signal in a paracrine manner to boost fungicidal activity following secondary challenge in the and lung (97, 98). Murine Ly6Chi represent effector cells against C. albicans (99), A. fumigatus (100), and B. dermatiditis (101). During repetitive challenges with β-glucans, murine Ly6Chi monocytes and human CD14+ monocytes exhibit attributes reminiscent of immunologic memory, including enhanced respon- siveness, based on cytokine responses (102). β-Glucan priming induces epigenetic and metabolic chang- es in monocytes, the latter of which associates with a shift from oxidative phosphorylation to glycolysis via a dectin-1/AKT/mTOR/HIF-1α signaling pathway (103) and increased glutaminolysis (104). In macrophages, β-glucan priming partially reverses LPS exposure–associated chromatin modifications, specifically the silencing of proinflammatory genes (105). In humans, a recent study proposes a role for STAT1 signaling in eliciting trained immunity, and by extension, in protection against chronic mucocu- taneous candidiasis (CMC) (106). Adaptive antifungal immunity. Antibody-mediated immunity to fungal pathogens has been extensive- ly reported in the literature (107). For example, natural fungal polysaccharide–targeting IgM antibodies enhance DC-mediated recognition of fungal antigen, the development of Th2 and Th17 responses, and B cell isotype class-switch recombination during murine pneumocystosis (108). In a model of X-linked agammaglobulinemia, pulmonary and CNS cryptococcal disease progressed rapidly, in part due to IgM-dependent defects in macrophage phagocytosis (109). Although there is significant support for the concept that antibody-dependent opsonization and complement activation enhance fungal clearance in animal models (107), loss of antibody-mediated immunity is generally compensated by alternate effec- tor systems, such as myeloid and CD4+ T cell–mediated immunity. Additionally, humans with humoral immune defects are generally not susceptible to fungal disease; however, protective fungus-specific anti- bodies are being researched as an adjunctive therapy in preclinical models (107). Several groups have generated protective antibodies that form the basis for vaccine strategies in murine models of fungal disease and in a phase I trial for recurrent vulvovaginal candidiasis (110–113) (NIH ClinicalTrials.gov identifier: NCT01067131). insight.jci.org https://doi.org/10.1172/jci.insight.93156 7 REVIEW

In the 1980s and 1990s, the dominant CD4+ T cell–dependent protection model involved the dichot- omous differentiation of CD4+ T cells into Th1 and Th2 subsets. More recent studies have shown that antibody- or cytokine-mediated disruption of Th1 immunity or Th2-favoring interventions correlate with adverse outcomes in murine fungal infection models (5, 9). The discovery of Th17 and regulatory T cells (Tregs), the development of CD4+ T cell receptor–transgenic mice (114–116), the identification of fungal epitopes that elicit antigen-specific responses (117–119), and the precise phenotyping of PIDD patients with mycoses have advanced insight into CD4+ T cell–driven antifungal immunity (120). At the site of infection, fungal cells and antigens are internalized by tissue-resident DC subsets and -derived cells (Mo-DCs). In the lung, Ly6Chi monocytes and derivative Mo-DCs transport A. fumigatus conidia and B. dermatiditis yeast cells to lung-draining lymph nodes (121, 122). In a B. derma- tiditis vaccine model, fungal protease–dependent cleavage of CCR2 ligands impedes lung Ly6Chi monocyte trafficking and the development of vaccine protection (123). However, Mo-DCs are not required for direct CD4+ T cell priming due to antigen transfer to –resident DCs (122). Mo-DCs, as well as IRF4- dependent CD11b+ DCs, instruct fungal antigen–specific Th17 responses followingA . fumigatus conidial challenge (124, 125). In the oral mucosa, CCR7-dependent transport of candidal antigens to draining lymph nodes results in antigen presentation by FLT3L-dependent migratory DCs and by Mo-DCs, both of which have the capacity to prime antigen-specific CD4+ T cells (116). In the skin, epidermal Langerhans cells recognize C. albicans yeast cell β-glucan via dectin-1, release IL-6, and instruct Th17 cell differentiation. These Th17 cells protect against secondary skin infection, but not against secondary systemic challenge (126). When C. albicans pseudophyphae penetrate the epi- dermis, dermal CD11b–CD103+ DCs drive differentiation of Th1 cells (126), which protect against sec- ondary systemic challenge, but not secondary skin challenge (126). Thus, the cellular requirements for CD4+ T cell priming and differentiation vary by anatomic site and fungal morphology, and, in most cases, involve antigen transfer from migratory DCs and Mo-DCs to lymph node–resident DCs. During pulmonary A. fumigatus challenge, differentiation occurs incrementally. Expression of the transcription factor T-bet, which controls Th1 differentiation, is detectable in antigen-specific CD4+ T cells in lung-draining lymph nodes and is enhanced by MyD88-dependent signals. In infected airways, MyD88- independent signals promote further Th1 differentiation and IFN-γ production (114). In this model, dectin-1 signaling counters Th1 differentiation by limiting innate IL-12p35 and IFN-γ production (124). Vaccine immu- nity to dimorphic fungi relies primarily on pulmonary Th17 cells that likely facilitate mononuclear phagocyte– and neutrophil-dependent killing of these pathogens (127, 128). Recently, researchers identified a fungal cal- nexin epitope that is widely conserved among ascomycetes (118). Vaccine delivery of fungal calnexin elicited calnexin-specific CD4+ T cells that conferred protection against the three North American dimorphic fungal pathogens (118). Calnexin represents the most promising candidate for vaccine design against these mycoses. Protective Th17 responses at mucosal surfaces can be enhanced by Treg-mediated IL-2 consumption, which enhances IL-17 and IL-22 release by responding Th17 cells and increases resistance to mucosal fungal infection (129). Despite the obligate requirement for IL-17 signaling during systemic candidiasis (61), FOXP3+ Treg–mediated potentiation of Th17 responses appears detrimental (130), consistent with the notion that high IL-17 levels lead to immunopathology, as described for type I IFN (38) and CCR1 signaling (131). During pulmonary cryptococcosis, mammalian chitotriosidase digests fungal chitin, thereby stimulating lung-resident CD11b+IRF4+ DCs, which in turn promote a Th2-dominant response to virulent serotype A strains (119). Compared with WT mice, C. neoformans challenge in chitotriosidase-deficient animals elicited dramatically lower antigen-specific Th2 cell numbers and extended murine survival, despite similar lung fungal burden (119). These data support a model in which antigen-specific Th2 cells exacerbate cryptococcosis by augmenting tissue damage without effecting fungal growth. Consistent with these findings, chitosan-deficient (i.e., deacetylated chitin) cryptococcal strains elicit cytokine mediators that promote a Th1-biased response (132). Similarly, the chitin content of individual A. fumigatus strains correlates with the magnitude of Th2 responses and lung eosinophil recruitment (133). The role of eosinophils in fungal clearance remains poorly understood, with several recent reports suggesting these cells help mediate fungal clearance (134–136).

Antifungal immunity: lessons from fungal disease–associated Mendelian disorders In the 1970s–1980s, characterization of two prototypic PIDDs that result from phagocyte oxidative machinery defects — CGD, caused by mutations in NADPH oxidase subunits and myeloperoxidase (MPO) deficiency insight.jci.org https://doi.org/10.1172/jci.insight.93156 8 REVIEW

(137, 138) — catalyzed our recognition that phagocyte oxidative cytotoxicity is critical for protection against invasive fungal disease in humans. The spectrum of clinical phenotypes of these disorders highlights the fungus-specific dependence on oxidative versus nonoxidative cytotoxic mechanisms for defense. For example, pulmonary aspergillosis is the signature fungal infection in CGD (137). Yet, only ~40% of CGD patients develop aspergillosis during their lifetime, despite daily inhalation of ubiquitous conidia. This observation indicates that nonoxidative mechanisms compensate in the remainder of the Aspergillus-exposed patients; better understanding of these mechanisms is an important future research direction (9, 69, 71, 77, 139). Inva- sive infections by other molds, such as Mucorales and Fusarium, or by commensal Candida yeasts are very uncommon (<5%), whereas invasive infections by endemic dimorphic fungi and Cryptococcus, or CMC do not develop in CGD (137, 140). In contrast, MPO deficiency is not permissive to infections by inhaled molds, and only a minority (<5%) of MPO-deficient patients develops invasive candidiasis. These clinical observations show that human phagocytes differentially depend on their oxidative capacity to effectively control different fungal pathogens. Importantly, while both CGD and MPO-deficient patients manifest defective hypochlorous acid production within phagocytes (137, 138), they exhibit distinct features in the phenotypic expression of fungal disease. The discrepancy in susceptibility to mold infection in these patients may reflect, at least in part, a potentially significant contribution of superoxide anion–dependent +K influx in activating phagolysosomal granule proteases (141), which is defective with NAPDH oxidase, but not MPO, deficiency. In the 1990s–2000s, the discovery of PIDDs resulting from alterations of IL-12/IFN-γ signaling uncovered the critical role of IL-12/IFN-γ–dependent lymphocyte/macrophage crosstalk in control of intracellular pathogens, including endemic dimorphic fungi, Cryptococcus, mycobacteria, and Salmonella (65). This pathway is dispensable for control of inhaled molds and mucosal fungal infection in humans. Some of these disorders respond clinically to mechanism-based immunotherapy with IFN-γ or IFN-α (142). Similarly, neutralizing IFN-γ and GM-CSF autoantibodies can underlie adult-onset acquired immunodeficiency characterized by endemic dimorphic fungal (143) and CNS cryptococcal disease (144). Patients with alveolar proteinosis due to impaired GM-CSF signaling develop aspergillosis (145), consis- tent with murine studies (146). During the last decade, careful clinical phenotyping combined with next-generation sequencing tech- nologies revealed the critical balance of JAK/STAT signaling in human antifungal immunity (147). Patients with STAT1 gain-of-function (GOF) mutations and those with hyper-IgE syndrome (Job’s syndrome) due to STAT3 loss-of-function (LOF) mutations develop fungal disease with overlapping and distinct features. STAT1 GOF mutations lead to disseminated infections by intracellular dimorphic fungi, CMC, and/or infections by inhaled molds in the absence of structural lung disease (65, 148). STAT3 LOF mutations also result in CMC, but pulmonary infections by inhaled molds occur as a consequence of structural lung disease caused by prior bacterial lung infections. In this cohort, infections by intracellular dimorphic fungi occur very rarely and, when they do, tend to involve the (149). In contrast, LOF STAT1 mutations or GOF STAT3 mutations do not cause fungal disease (65). Mutations in the transcription factor GATA2 result in a protean PIDD (65, 150) that brings together (a) susceptibility to bacterial, fungal and , (b) malignancy, and (c) vascular abnormalities. From a fungal disease standpoint, GATA2 haploinsufficiency leads to infections by endemic dimorphic fungi,Cryp - tococcus, and Aspergillus, but not Candida. GATA2-deficient patients exhibit monocytopenia that, consistent with recent murine models, likely contributes to fungal susceptibility (100, 150). The discovery of kindreds with CMC that carry mutations in IL17F, IL17RA, IL17RC, and NF-κB activator 1 (ACT1), and complementary studies in IL-17–mutant mice discussed above indicate that IL-17 signaling is indispensable for mucosal, but not systemic, antifungal immunity (64, 65). Patients with IL17F or IL17RC mutations only develop CMC, whereas those with IL17RA or ACT1 mutations also manifest cutaneous staphylococcal and/or pulmonary bacterial infections, indicating that IL-17E/IL-25 signaling may specifically map to bacterial defense at mucosal surfaces (151, 152). CMC susceptibility has also been linked to mutations in other genes (64, 65), most of which either directly or indirectly interfere with Th17 development and/or responses (Figure 2). These include AIRE deficiency in autoimmune polyendocri- nopathy-candidiasis-ectodermal dystrophy (APECED) (153), Job’s syndrome in which STAT3 regulates RORγt-dependent Th17 development, the various genetic forms of severe combined immunodeficiency disorder (154), and mutations in STAT1, RORC, STK4, IRF8, DOCK8, IKBA, CLEC7A, and CARD9 (64, 65, 155). A CLEC7A polymorphism is also associated with susceptibility to invasive aspergillosis in hematopoi- etic cell transplant recipients (156). insight.jci.org https://doi.org/10.1172/jci.insight.93156 9 REVIEW

CARD9 deficiency exhibits three distinctive features among Mendelian disorders of antifungal immunity (157). First, a substantial proportion of CARD9-deficient patients exhibit adult-onset fungal disease, in contrast to early childhood onset typical of other PIDDs. It is unclear whether a different threshold of fungal exposure and/or additional genetic, environmental, or other factors accounts for this observation. Second, CARD9 deficiency is the only PIDD in which both mucosal and systemic fungal disease develop, and susceptibility is restricted to fungi without concurrent bacterial or viral infections. , CMC, invasive , and superficial and deep-seated dermatophy- tosis have also been reported (157). In contrast to CARD9, MALT1 or BCL-10 deficiency results in a narrower spectrum of fungal susceptibility, with only mucosal infections reported to date (157). Third, CARD9 deficiency strikingly results in infections at anatomical sites that are not typically associated with the specific pathogens. For example, CARD9-deficient patients exhibit a predilection forCandida meningoencephalitis, without disease in kidney, liver, or spleen, all of which are commonly affected in CARD9-sufficient patients (157, 158). CARD9 is critical for neutrophil recruitment to theCandida - infected CNS but not for neutrophil recruitment to the Candida-infected kidney or Staphylococcus-infected CNS (159). This tropism is mediated by CARD9-dependent production of CXC chemokines by resi- dent glial cells and neutrophils in the Candida-infected CNS, while neutrophil-intrinsic chemotaxis and effector functions are largely intact in CARD9-deficient humans and mice (159–161). Development of CNS and intra-abdominal aspergillosis is another example of altered tissue tropism in CARD9-deficient patients (162) and results from defective CARD9-dependent neutrophil chemoattractant production in and neutrophil mobilization to extrapulmonary infection sites. The absence of pulmonary Aspergillus involvement in CARD9 deficiency may be explained by compensatory IL-1R/MyD88–dependent neu- trophil lung recruitment (161, 163). Collectively, these human observations have led to our appreciation that CARD9 appears to mediate fungus-, cell type–, and organ-specific neutrophil recruitment during invasive fungal infection. GM-CSF and G-CSF were reported to improve the outcome of a small number of CARD9-hypo- morphic patients with CNS candidiasis (164). In this cohort, mutant CARD9 was impaired in its ability to complex with RASGRF1, but not with BCL10 and MALT1 (165). The mechanism by which these cyto- kines bypass the CARD9-dependent immune defect in this cohort and the generalizability of this protec- tion in other CARD9-deficient patients remain to be elucidated.

Antifungal immunity and the study of the mycobiome While commensal fungi have been noted in humans and mice for over five decades, lack of culture-indepen- dent methods, delay in developing high-throughput rDNA sequencing methods, and paucity of annotated reference databases to classify fungal rDNA amplicons delayed characterization of endogenous fungal communities, termed the (166, 167). Although fungi comprise less than 1% of total microbial rDNA sequences at different anatomic sites, the size of fungal cells (~3–10 μm diameter for most yeast cells, 2–5 μm diameter for typical mold conidia, and 10 to hundreds μm length for hyphae, compared with < 1 μm diameter for bacterial cells) suggests that fungal rDNA quantification underestimates fungal bio- mass in the microbiota. In the past five years, researchers have analyzed endogenous fungal communities in the oral cavity, gastrointestinal tract, skin, and mucosal sites in healthy and diseased individuals (168–172). In most studies, between 15 and 70 fungal genera have been identified, withMalassezia spp. predominant in the skin and Candida spp. predominant in the intestine. Recent work has defined mechanisms ofCandida colonization resistance by intestinal anaerobic (173). Our understanding of the reciprocal interplay between endogenous fungal communities and anti- fungal immunity remains limited. Despite high-quality studies that point to a central role for IL-17 in mucosal antifungal immunity, it remains unclear whether IL-17 controls the composition or diver- sity of commensal fungi. Administration of secukinumab, an IL-17–neutralizing Ab, in patients with Crohn’s disease increased the rate of fungal infections, consistent with IL-17 directing mucosal antifun- gal immunity (174). Recent work has examined dectin-1 in shaping endogenous fungal communities and mycobiota-trig- gered immune responses in the gut. Clec7a–/– mice developed intestinal inflammation andCandida and spp. overgrowth in the gut (168). In turn, Clec7a–/– mice were unable to control Candida during dextran sulfate sodium–induced colitis and benefited from therapy. In the absence of commen- sal Candida, Clec7a–/– mice were more resistant to colitis and exhibited a reduction in colonic antimicrobial insight.jci.org https://doi.org/10.1172/jci.insight.93156 10 REVIEW

peptides that target Gram-positive bacteria, leading to an increase in commensal Lactobacillus murinus bacte- ria, Treg expansion, and resistance to colitis (175). Colonization of Clec7a–/– mice with C. tropicalis reversed these effects and promoted intestinal inflammation. These findings suggest that dectin-1 signaling indirectly influences the intestinal bacterial microbiota. In humans, a two-markerCLEC7A haplotype is associated with treatment-refractory ulcerative colitis (168). Another study identified a negative correlation between a CLEC7A single nucleotide polymorphism (SNP) and Malassezia sympodialis abundance in the gut of inflam- matory bowel disease (IBD) patients, though no additional (positive or negative) correlations with other fungi were reported, likely due to the small cohort size (176). These studies highlight a protective role for dectin-1 in intestinal mycobiota control and antifungal immunity. Dectin-3 was recently reported to contribute to control of intestinal Candida and colitis. Intestinal C. tropicalis overgrowth in Clec4d–/– mice was accompanied by a decrease in Th17 cells, impaired macrophage fungal phagocytosis, and defective intestinal epithelial cell barrier function (177). Consistent with these findings, antifungal treatment ofClec4d –/– mice reduced C. tropicalis burden and ameliorated colitis. CARD9 has also been implicated in IBD pathogenesis, as a nonsynonymous SNP in the CARD9-cod- ing region strongly associates with IBD risk (178). In experimental colitis models, CARD9 signaling can be protective against fungi that contact the intestinal mucosa during colitis. Furthermore, antifungal drugs partially ameliorate intestinal inflammation in this context (179). Analysis of the intestinal microbiota of Card9–/– mice revealed alteration in fungal and bacterial communities compared with WT animals. The intestinal microbiota in Card9–/– mice lacked tryptophan-metabolizing bacteria and did not produce aryl hydrocarbon receptor ligands (180). As a result, Card9–/– mice showed defective expression of Il22 and anti- microbial peptide–encoding genes Reg3g and Reg3b. Altogether, these findings suggest CARD9 may dually control fungal and bacterial populations in the gut. Immunologic effects of intestinal fungi extend beyond the gut. In a house dust mite–induced airway allergy model, disruption of the gut fungal community with antifungal drugs increased disease severity (181). Aspergillus amstelodami, Epicoccum nigrum, and increased in abundance during antifungal drug–induced fungal dysbiosis, and intestinal supplementation with these strains replicated the detrimental effects of antifungal drugs on lung allergy (181). Similarly, -induced fungal overgrowth exacerbat- ed papain-induced lung allergy by promoting intestinal Candida overgrowth (182). This phenomenon, first demonstrated with C. albicans (183), is recapitulated by multiple Candida species and is mediated in part by eicosanoids (182). Thus, there is a complex interplay between endogenous fungal communities and innate immune tone and responses, both at local and distant sites.

Conclusions and future perspectives The field of antifungal immunity has rapidly advanced in the past decade, a period marked by the dissec- tion of fungus-specific innate and adaptive immune responses and convergence of human clinical and ani- mal model data. The advances during this era are exemplified by insights into the pivotal role of dectin-1/ CARD9 and IL-17 pathways in antifungal immunity. The discovery of β-glucan–induced trained immunity and conserved sterilizing immunity-mediating epitopes lays the foundation for clinical trials to test vaccine protection against multiple fungal genera and species. Important areas of future research include eluci- dation of the role of epithelial surfaces in fungal virulence and antifungal defense and the intercellular crosstalk underlying innate and adaptive antifungal immunity. It is likely that additional fungal recognition receptors and response pathways remain to be discovered, both in animal models and in human genetic studies. How the composition, diversity, and metabolism of endogenous fungal communities contribute to immune homeostasis and to inflammatory disorders remains largely unexplored, yet are central for deci- phering the contribution of fungi to diverse states of human health and disease.

Acknowledgments The authors receive support from the Division of Intramural Research of the National Institute of Allergy and Infectious Diseases (MSL), NIH grants AI093808 (TMH), DK098310 (IDI), AI123819 (IDI), a Ken- neth Rainin Foundation Award (IDI), a Burroughs Wellcome Fund Investigator in the Pathogenesis of Infectious Diseases Award (TMH), and a Geoffrey Beene Foundation Award (TMH). NIH Core Grant P30 CA008748 to MSKCC provided support for this manuscript. The authors apologize to many contribu- tors in the field whose work could not be cited due to space limitations.

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Address correspondence to: Michail S. Lionakis, National Institutes of Health, 9000 Rockville Pike, Build- ing 10, Room 11C102, Bethesda, Maryland, 20892, USA. Phone: 301.443.5089; E-mail: lionakism@ niaid.nih.gov. Or to: Iliyan D. Iliev, Weill Cornell Medicine, Belfer Research Building, Room BB 718, 413 East 69th Street, New York, New York 10021, USA. Phone: 646.962.7236; E-mail: [email protected]. edu. Or to: Tobias M. Hohl, Memorial Sloan Kettering Cancer Center, 1275 York Avenue, Box 9, New York, New York, 10065, USA. Phone: 646.888.3596; E-mail: [email protected].

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